Serum-free cell culture media

ABSTRACT

A serum-less medium for use in the growth of cells. The medium is an improved aqueous culture media of the MEM type comprising thyroxin, insulin, and Vitamin A. Hydrocortisone and essential fatty acids may also be advantageously used, as may effective quantities of Vitamin E. Surfactants are used to make the vitamins and any fatty acids available for cell growth. This aqueous media may also comprise cell-growth-enhancing quantities of biotin and folic acids.

RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.951,500 filed Oct. 6, 1978 which was a continuation-in-part ofapplication Ser. No. 866,338 filed Jan. 1, 1978, both now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a novel cell growing medium and novel processfor growing living cells.

Over the years there has been a substantial amount of effort directed tothe problem of providing a versatile and efficacious culture media fromreproducible, fully-characterized components. This "ideal" approach isfrequently compromised with respect to the "fully characterized" natureof the components. For example, much work has been done (e.g. asdescribed in U.S. Pat. Nos. 3,128,228 by J. Michl and 3,953,290 by K.Uthne) describing the use of serum of blood fractions in cell culturemedia. However, these fractions have some of the disadvantages of wholeserum and other bulk protein sources. They are not as fullycharacterized as is desirable; that is, they will differ from batch tobatch and introduce unknown variables in the media. Such variables canaffect the growth of cells and can do so in wholly umpredictable ways.

Other workers have recognized the value of culture media which is freeof serum and other bulk protein supplements which can interfere withcell replication. To date, some of these attempts have proved partiallysuccessful. Although the media developed in each case has seriouslimitations.

Thus, Torney et al in U.S. Pat. No. 3,887,930 and U.S. Pat. No.4,055,460 describes a media containing a particulate resin whichprovides an undesirable substrate on which cells can grow and from whichtheir removal can be difficult.

The work described by Bower, Arthur and Fine, Propagation of mousemammary tumor cell lines and production of mouse mammary tumor virus ina serum free media, in In Vitro (Pages 558-563, Volume 12, No. 8, 1976),utilizes a media containing considerable amounts of ethyl alcohol andlipids. The high volatility of ethyl alcohol (much higher than that ofthe acqueous media), which is used in this media to disperse the lipidsin the acqueous solution, predisposes their media to serious problems.Gradual evaporation of the alcohol must be expected in the conditions tobe found in cell culture incubators (about 37° C. with a variable CO₂concentration in the atmosphere), causing their media to separate into abiphasic (lipid-acqueous) mixture. This type of mixture would not beappropriate for most cell culture work, nor would its exact compositionbe easily regulated. Besides, high concentration of alcohol, by itself,may have undesirable effects upon cell growth in many situations.

Other work, as described by S. T. Donta in "The growth of functional ratglial cells in a serumless medium." in Experimental Cell Research (Pages119-124, Volume 82, 1973), has succeeded in adapting a single,specialized cell line to grow in a chemically defined media without bulkprotein supplementation. However, this media has been unable to supportthe growth of other cell lines, unless used in combination with albumin.Thus, the media is not versatile unless supplemented with proteins whichare not fully characterized.

Still another work, as described in U.S. Pat. No. 4,049,494, relates toprocesses for growing cells in serum-less media wherein the cells arefrom cell lines which have been specially adapted to function in themedia. Such processes and media, of course, are lacking in broadutility. U.S. Pat. No. 4,072,565 describes the use of protamine zincinsulin in processes limited to short-terms by limitations inherent inthe media selected.

Therefore, even after one decides on the ideal of providing a serum-freemedia, it is a further problem to provide a media with a combination ofnutrients versatile with respect to a cell type that can be grown in it.Also, these nutrients, once identified, must be made available to thecells in a controlled fashion, without the introduction of highlyvolatile components of biphasic mixtures of variable composition.Furthermore, such a media must be free of particulate elements whichwould introduce unwarranted growth surfaces. Finally, this media shouldnot be supplemented with partially refined bulk protein sources.

SUMMARY OF THE INVENTION

It is a principal object of the invention to provide an improved aqueousculture medium for growing cells.

It is a particular object of the invention to provide a culture mediumthat is fully characterized and, consequently, subject to accuratereplication without dependence on animal blood serums or on othernon-characterized materials, e.g. the bulk protein supplements commonlyused in the art.

A further object of the invention is to provide a culture mediadescribed in the foregoing objects which comprises specific combinationsof vitamins, fatty acids, and hormones to enhance cellular growth andwhich is substantially free of volatile or particulate components.

Still another object of the invention is to provide an improved basicculture media, one which is not necessarily optimized with respect tospecific nutrients, but which is a substantial improvement over minimumessential media (MEM), including those of the zinc option type, known tothe art.

A further object of the invention is to provide cell growth media ofimproved shelf life and versatility.

Another object of the invention is to provide an improved processutilizing the valuable features of the culture media of the invention.

Further objects of the invention include providing specific combinationsof cell-growth materials in such a way as to make them readily availableto growing cells over an extended period of time.

Other objects of the invention will be obvious to those skilled in theart on their reading this disclosure.

The above objects have been substantially achieved by (a) thedevelopment of an improved basic culture media of the MEM-zinc optiontype. In the more advantageous forms of the invention, an improved basicmedia is enhanced with hormones such as thyroxine, and insulin andVitamin A. It is also advantageous to add hydrocortisone and essentialfatty acids such as linoleic, linolenic and arachidonic acids. Vitamin Eis also a desirable additive. In each case an acceptablywater-dispersable, physiologically-equivalent compound such asphysiologically equivalent salts, isomers, homologues, polymers, orother derivatives may be utilized. In general, the physiologicalequivalents are well known to the art.

It is to be emphasized that the process and media of the instantinvention is useful with a wide variety of cells without specialselective breeding products to build up a special adaption to the serum.Moreover, the cell growth can be prolonged over periods of three monthsor more in many advantageous modes of the invention.

The Basic Medium

One basic medium, found advantageous by itself and also advantageous foruse as a base for a selective augmentation, is formed with substantialquantities of amino acids, biotin and folic acid. This basic medium,fortified to achieve an improved amino acid profile and fortified withthe indicated vitamins, provides a growth culture media of improvedversatility and performance.

To form the preferred growth media of the invention, the following areto be added to the fortified basic medium as described above.

Thyroxine: preferably T₄ but T₃ could also be used. The material isavailable in many commercial forms, including salt forms. Quantities ofT₄ from 0.01 to 0.03 micrograms per liter are desirable.

Insulin: A quantity of about 4 milligrams per liter is used.

Hydrocortsone: Again, this material is available in many forms. What isessential is to have a cortisone-active nucleus. A quantity of fromabout 0.075 to 0.3 milligrams per liter is preferred.

Essential Fatty Acids: The preferred acids are linoleic, linolenic andarachidonic. Although not water soluble, these materials are soemulsified with an appropriate surfactant as to become distributed inthe aqueous medium in such a way that their molecules are readilyavailable to the cell-growing population even in the preferred aqueousgrowth media. A quantity of from about 2.5 to 15 milligrams per liter isrequired.

Vitamin E and Vitamin A: Vitamin A is preferably used in a quantity offrom about 0.1 to 0.4 milligrams per liter. Vitamin E is preferably usedin quantities of from about 7.5 to 30 milligrams per liter.

Surfactants: In a sufficient quantity to disperse the Vitamins and fattyacids and, thus, provides means to assure availability of the fattyacids and vitamins to the growing cells. A polysorbate 20 isadvantageously used.

It is presently believed that the thyroxin, insulin and Vitamin A shouldbe utilized in all embodiments of the invention. Hydrocortisone andessential fatty acids are highly advantageous. Vitamin E is believed tobe important in contributing to the versatility of the invention.

One of the problems with developing a serumless tissue culture mediacomes with passaging, e.g. transferring, the cells between vessels.Proteolytic enzymes (trypsin or the like) will not be neutralized bythis kind of media, since a factor present in serum is active in theneutralization process, and can result in damage or death to the cells.Appropriate physical or mechanical means to detach and passage the cellsare, therefore, preferable. If proteolytic enzymes become necessary forwhatever reason for effective cell detachment, it is suggested that thecells should be washed before replating in media containing enzymeinhibitors which are commercially available. In any event, it isdesirable to avoid cell-damaging quantities of enzymes like trypsin ineffecting transfer of these serum-free cell populations.

It is to be understood that the various components of the growth mediaof the invention can be obtained from natural sources when it isconvenient to do so. For example, the fatty acids could be madeavailable as processed vegetable oils, e.g. soybean or safflower oil. Insuch cases, it is necessary to so process the natural material as toremove any substantial quantities of unknown proteinaceous substances.

Although, it is usually preferable to use a zinc-option-type minimumessential medium (MEM) it is possible to achieve a substantial number ofthe advantages using the invention by appropriate modification of suchcommercial products known to those skilled in the art, such as Medium199 Eagle's Basal Medium, Eagle's Minimum Essential Medium or the like.

Illustrative Examples of the Invention

It is, of course, to be understood that the following examples areintended to be illustrative and that numerous changes can be made in thereactants, precise proportions, and conditions set forth therein withoutdeparting from the spirit of the invention as defined in the appendedclaims.

A fortified basic medium formula is prepared according to goodpreparatory procedures as known in the art.

    ______________________________________                                        Item          Amount (milligrams per liter)                                   ______________________________________                                        L-alanine     8.9                                                             L-asparagine  75.0                                                            L-aspartic acid                                                                             13.0                                                            L-glutamic acid                                                                             14.7                                                            L-glycine     7.5                                                             L-proline     11.5                                                            L-serine      52.5                                                            L-arginine HCl                                                                              196.52                                                          L-cystine     37.2                                                            L-glutamine   584.0                                                           L-histidine HCl                                                                             65.0                                                            L-isoleucine  80.88                                                           L-leucine     160.88                                                          L-lysine      112.38                                                          L-methionine  23.30                                                           L-phenyl-alanine                                                                            50.15                                                           L-threonine   74.18                                                           L-tryptophan  15.61                                                           L-valine      71.4                                                            L-tyrosine    45.9                                                            CaCl          200                                                             KCl           400                                                             .[.MgCl6H.sub.2 O.].                                                          .Iadd.MgCl.sub.2 . 6H.sub.2 O.Iaddend.                                                      183                                                             MgSO.sub.4 7H.sub.2 O                                                                       12                                                              NaCl          6800                                                            NaH.sub.2 PO.sub.4 H.sub.2 O                                                                150                                                             NaHCO.sub.3   sufficient to adjust to a Ph of 7.4                             phenol red    10                                                              glucose       2000                                                            Na pyruvate   110                                                             biotin        1.0                                                             D-calcium pantothenate                                                                      1.0                                                             choline Cl    56.0                                                            folic acid    1.0                                                             inositol      36.0                                                            nicotinamide  1.0                                                             pyridoxal HCl 1.0                                                             riboflavin    0.1                                                             thiamine      1.0                                                             B.sub.12      1.36                                                            folinic acid  1.0                                                             DL thioctic acid                                                                            0.20                                                            putrescine HCl                                                                              0.16                                                            linoleic      0.084                                                           FeCl.sub.3 6H.sub.2 O                                                                       0.54                                                            ZnSO.sub.4 7H.sub.2 O                                                                       0.14                                                            insulin       4.00                                                            HEPES buffer solution                                                                       2380.0                                                          ______________________________________                                         Distilled water to make a total of 1 liter                               

In general, the quantities of ingredients set out above can be modifiedby about plus-or-minus 20%; which modifications are to be construed tobe "about" those required to function as does the listed formula.

HEPES buffer solution is the name given to buffer solutions containingN-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid. It has a pK_(a)value of about 7.5.

The aforesaid basic media was used in Example 1 set forth below.

EXAMPLE 1

Table 1C illustrates the advantageous characteristics of this basicmedium when it is compared to

A. the improved fortified basic media of the invention both with andwithout the addition of 10% fetal calf serum;

B. a commerically available zinc-option-bearing MEM formula sold underthe trade designation IMEMZO & HEPES by Associated Biomedic Systems,Inc., of Buffalo, N.Y. Again, the comparison was carried out with andwithout augmentation of the formula with 10% fetal calf serum.

The drawing is indicative of the cell growth in terms of thimidineradioactive count (TRC) as follows:

                  TABLE 1C                                                        ______________________________________                                        Growth Media and Cell History                                                                            Count                                              ______________________________________                                        IMEMZO & HEPES, no serum-cell line                                                                       nil                                                IMEMZO & HEPES, no serum initial culture                                                                 nil                                                Basic Media of Invention, no serum-initial culture                                                        1,533                                             Basic Media of Invention, no serum-cell line culture                                                     54,672                                             Basic Media of Invention, serum-cell line culture                                                        20,825                                             Basic Media of Invention, serum-initial culture                                                           8,293                                             ______________________________________                                    

"Initial culture" is used to define the primary tumor cells. "Cell line"is used to define cells grown on tissue culture flask for a minimum offive passages.

It will be readily seen that the Basic Media of the invention issuperior in cell-line culturing procedures, not only to media withdifferent fully-characterized profiles (IMEMZO) but to itself when fetalcalf serum is incorporated therein.

The results disclosed in Example 1 were obtained according to thefollowing procedure:

The tumor cells used in this experiment were rhabdomyosarcoma of mice,Balb-C strain, produced in the laboratory by the intramuscular injectionof methylcholanthrene in experimental animals. The tumor was observed toform within several weeks of injection. This tumor was subcultured andkept by serial passage within a line of Balb-C mice and in serumsupplemented tissue culture media by different researchers.

A suspension of viable cells were injected subcutaneously in Balb-C mice(from Charles River Breeding Labs) in a concentration of 1,000,000viable cells per injection. Tumor growth was carefully monitored inthese mice.

Cells obtained from these tumors may be classified in several differentfashions. "Primary tumor cells" define cells obtained by the disperionof the tumor grown anew, fresh, in a Balb-C mouse. These cells wereseparated from the tumor, by the use of standard trypsinizationtechniques. Trypsin was neutralized with serum and the cells were washedbefore further use. Their viability was assayed by the trypan bluetechnique. These cells are labelled as "Primary Tumor Cells."

Cell lines were cultured in tissue culture media from these primarytumor cells by using basic media, as described in the body of thisdisclosure, e.g. Example 1. However, this basic media used to establishthe cell lines contained 10% (by volume) of fetal calf serum. Prior touse of these cell lines, the cells were again dispersed by thecold-media technique described below and their viability checked by thetrypan blue technique. These cells are referred to herein as "celllines."

Transferring of the cells between vessels, in multipassages work isaccomplished by adding cold media (about 4° C.) to the flask followed byshaking and dispersion by vortexing. In this way the cells are coldshocked and readily released for transfer .[.way the cells are coldshocked and readily released for transfer .]. to a new vessel and a newpassage

The following procedure is used to test for the activity of thedifferent growth media formulations:

The growth of cells was estimated by the amount of incorporation ofradioactively labelled (tritium labelled) thimidine. Thimidine isincorporated by growing cells for the manufacture of DNA. Thus, theamount of radioactivity incorporated into growing cells incubated inradiactively labelled thimidine directly approximates the growth thathas taken place within the cell culture. Cells were grown in thisfashion using each of stock-IMEMZO and the basic medium of theinvention. For each medium, experiments were carried out with mediacontaining 10% (by volume) fetal calf serum and media containing noserum.

Incorporation of radioactively labelled thimidine by primary tumor cellsand cell lines grown in stock-IMEMZO media either with or without fetalcalf serum supplementation, is nil or not significantly different thanthe background radioactive count. Primary cells grown in basic mediawith no serum supplementation shows an incorporation of 1,533radioactive counts. Primary cells grown in basic media with 10% fetalcalf serum supplementation showed an incorporation of 8,293 counts.

The cell line, as described above, is a primary tumor cell that has beenserially grown in vitro for a minimum of 5 passages. A passage beingdefined as that period of time it takes for the cells to form amonolayer. This is followed by dispersion of this monolayer andreculturing of cell aliguots in a fresh flask.

Testing of the cell lines resulted in markedly different results. Thecell line grown in stock-IMEMZO media with or without serumsupplementation again was unable to incorporate radioactivity to anyappreciable extent. The cell line grown in basic media with no serumsupplementation showed an incorporation of 54,672 counts on the average.The cell line grown in basic media supplemented with 10% fetal calfserum showed an incorporation of only 20,825 counts.

In all cases, the cells were incubated for three days at 37° C. in a 5%CO₂ atmosphere before they were exposed to radioactively labelledthimidine for 12 hours and then counted again. In each case,quadruplicate culutures of 10⁵ viable cells were carried out by use of amicrotiter plate culturing system. These cells were processed forcounting with an automatic harvesting machine and counted in a Searleliquid scintillation counter.

The numbers given for incorporation of radio active counts represent theaverage number of counts per minute per culture, counted by the liquidscintillation apparatus. All the cells present in each culture wereharvested and the total radioactive content of the complete populationof cells in each culture yielded the radioactive counts expressed aboveas an average. Each different type of culture (determined by the mediaused, the cell type and the presence or absence of fetal calf serum inthe media) yielded, therefore, its own average count.

The following conclusions may be derived from the experiments above:

1. Stock-IMEMZO, either with or without serum supplementation, is unableto support the growth of these cells in vitro.

2. Basic medium with a 10% fetal calf serum supplementation is betterthan the same media without serum supplementation in supporting thegrowth of primary cells in vitro.

3. Basic medium with no serum supplementation supports the in vitrogrowth of cell lines up to 250% better than basic medium with 10% serumsupplementation.

Basic medium without serum supplementation seems to be clearly superiorto the same medium with 10% fetal calf serum supplementation in growingcell lines in vitro. Thereby it provides a medium able to sustain celllines in vitro without the use of serum or other bulk biologicalproducts.

To test the abiliy of the basic medium to sustain the growth of celllines for extended periods of time, the cell lines described above weregrown in basic medium, free of serum supplementation, for a period offive months. The changeover to serumless medium was accomplishedgradually, over 2 weeks. The cells were observed to clump initially, butsoon afterwards they regained their normal morphology and were observedto form monolayers and grow as well as the control cell lines kept inmedia supplemented with 10% fetal calf serum. Thus, the basic medium wasclearly successful in supporting the growth of these cell lines. At thispoint it was decided to terminate these experiments withmethylcholanthrene induced rhabdomyosarcoma cell lines and to starttesting various other cell lines for their growth in this media.

EXAMPLE 2--The Growth Media

To the above basic media were added the following:

    ______________________________________                                        Item              Amount                                                      ______________________________________                                        thyroxine         0.02 micrograms per liter                                   insulin           4 milligrams per liter                                      hydrocortisone    .[.1.5.]. .Iadd.0.15.Iaddend.                                                 milligrams per liter                                        linoleic acid     5.0 milligrams per liter                                    linolenic acid    5.0 milligrams per liter                                    arachidonic acid  5.0 milligrams per liter                                    surfactant, emulsifier*                                                                         110 milligrams per liter                                    Vitamin E         20 milligrams per liter                                     Vitamin A         0.25 milligrams per liter                                   ______________________________________                                    

The surfactant is a micelle-forming material sold by Supelco Co. underthe trade designation Tween 20. The material is characterized by itsability to emulsify, or isolate and expose, molecules of fatty acids insmall micelle-type structures thereby making them readily dispersible inaqueous solutions and available to the growing cells. Any othersurfactant which achieves this effect with a reasonable concentrationand is not otherwise detrimental to cell growth may also be utilized.

The procedure of Example 1 is used to demonstrate the advantage of theabove-described culture media. Excellent results are achieved with celllines.

EXAMPLE 3

The following certified cell lines, all available in the American-typeCulture Collection at Rockville, Md., are also advantageously grown inthe growth medium of Example 2, and then counted according to theprocedure set forth therein:

MRC--Human embryonic lung cell line.

L929--Mouse fibrosarcoma line.

Vero--African green monkey kidney line.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed and all statements of the scope of the invention which mightbe said to fall therebetween.

What is claimed is:
 1. .[.An MEM zinc option.]. .Iadd.A cell culturing.Iaddend.medium wherein the amino acid profile is about as follows:

    ______________________________________                                        Item         Amount (milligrams per liter)                                    ______________________________________                                        L alanine    8.9                                                              L asparagine 75.0                                                             L aspartic acid                                                                            13.0                                                             L glutamic acid                                                                            14.7                                                             L glycine    7.5                                                              L proline    11.5                                                             L serine     52.5                                                             L arginine HCl                                                                             196.52                                                           L cystine    37.2                                                             L glutamine  584.0                                                            L histidine HCl                                                                            65.0                                                             L isoleucine 80.88                                                            L leucine    160.88                                                           L lysine     112.38                                                           L methionine 23.30                                                            L phenyl-alanine                                                                           50.15                                                            L threonine  74.18                                                            L tryptophan 15.61                                                            L valine     71.4                                                             L tyrosine   45.9                                                             ______________________________________                                    


2. .[.An MEM zinc option.]. .Iadd.A .Iaddend.medium, as defined in claim1, wherein said medium is augmented with cell growth promotingquantities of biotin and folic acid.
 3. A culture media as defined inclaim 1 further comprising

    ______________________________________                                        Thyroxine       0.01 to 0.03 micrograms per liter                             Insulin         4 milligrams per liter                                        Hydrocortisone  0.075 to 0.3 milligrams per liter                             Essential Fatty Acids                                                                         2.5 to 15 milligrams per liter                                Vitamin A       0.1 to 0.4 milligrams per liter                               Vitamin E       7.5 to 30 milligrams per liter                                Surfactants     Sufficient quantity to disperse                                               vitamins and fatty acids.                                     ______________________________________                                    


4. A culture media as defined in claim 2 further comprising

    ______________________________________                                        Thyroxine       0.01 to 0.03 micrograms per liter                             Insulin         4 milligrams per liter                                        Hydrocortisone  0.075 to 0.3 milligrams per liter                             Essential Fatty Acids                                                                         2.5 to 15 milligrams per liter                                Vitamin A       0.1 to 0.4 milligrams per liter                               Vitamin E       7.5 to 30 milligrams per liter                                Surfactants     Sufficient quantity to disperse                                               vitamins and fatty acids.                                     ______________________________________                                    


5. A culture media as defiined in claim 3 wherein said surfactant is apolysorbate
 20. 6. A culture media as defined in claim 1 furthercomprising

    ______________________________________                                        Item              Amount                                                      ______________________________________                                        thyroxine         0.02 micrograms per liter                                   insulin           4 milligrams per liter                                      hydrocortisone    .[.1.5.]. .Iadd.0.15.Iaddend.                                                 milligrams per liter                                        linoleic acid     5.0 milligrams per liter                                    linolenic acid    5.0 milligrams per liter                                    arachidonic acid  5.0 milligrams per liter                                    surfactant, emulsifier                                                                          110 milligrams per liter                                    Vitamin E         20 milligrams per liter                                     Vitamin A         0.25 milligrams per liter                                   ______________________________________                                    


7. An aqueous culture media for use in cell growth having about thefollowing formula:

    ______________________________________                                        Item          Amount (milligrams per liter)                                   ______________________________________                                        L alanine     8.9                                                             L asparagine  75.0                                                            L aspartic acid                                                                             13.0                                                            L glutamic acid                                                                             14.7                                                            L glycine     7.5                                                             L proline     11.5                                                            L serine      52.5                                                            L arginine HCl                                                                              196.52                                                          l cystine     37.2                                                            L glutamine   584.0                                                           L histidine HCl                                                                             65.0                                                            L isoleucine  80.88                                                           L leucine     160.88                                                          L lysine      112.38                                                          L methionine  23.30                                                           L phenyl-alanine                                                                            50.15                                                           L threonine   74.18                                                           L tryptophan  15.61                                                           L valine      71.4                                                            L tyrosine    45.9                                                            CaCl          200                                                             KCl           400                                                             .[.MgCl6H.sub.2 O.].                                                          .Iadd.MgCl.sub.2 . 6H.sub.2 O.Iaddend.                                                      183                                                             MgSO.sub.4 7H.sub.2 O                                                                       12                                                              NaCl          6800                                                            NaH.sub.2 PO.sub.4 H.sub.2 O                                                                150                                                             phenol red    10                                                              glucose       2000                                                            Na pyruvate   110                                                             biotin        1.0                                                             D-calcium pantothenate                                                                      1.0                                                             choline Cl    56.0                                                            folic acid    1.0                                                             inositol      36.0                                                            nicotinamide  1.0                                                             pyridoxal HCl 1.0                                                             riboflavin    0.1                                                             thiamine      1.0                                                             B.sub.12      1.36                                                            folinic acid  1.0                                                             DL thioctic acid                                                                            0.20                                                            putrescine HCl                                                                              0.16                                                            linoleic      0.084                                                           FeCl.sub.3 6H.sub.2 O                                                                       0.54                                                            ZnSO.sub.4 7H.sub.2 O                                                                       0.14                                                            insulin       4.00                                                            HEPES buffer solution                                                                       2380.0                                                          NaHCO.sub.3   sufficient to adjust to a pH of                                               about 7.4                                                       ______________________________________                                         Distilled water to make a total of 1 liter                               


8. A culture media as defined in claim 7 further comprising

    ______________________________________                                        Thyroxine       0.01 to 0.03 micrograms per liter                             Insulin         4 milligrams per liter                                        Hydrocortisone  0.075 to 0.3 milligrams per liter                             Essential Fatty Acids                                                                         2.5 to 15 milligrams per liter                                Vitamin A       0.1 to 0.4 milligrams per liter                               Vitamin E       7.5 to 30 milligrams per liter                                Surfactants     Sufficient quantity to disperse                                               vitamins and fatty acids.                                     ______________________________________                                    


9. A medium as defined in claims 3, 4, or 8 wherein said essential fattyacids are predominantly comprised of linoleic acid, linolenic acid andarachindonic acid.
 10. A medium as defined in claims 1, 2, 7, 3, 4, 8, 5or 6 wherein said medium is maintained substantially free ofcell-damaging quantities of proteolytic enzymes.
 11. An improved processfor growing lines of living cells comprising the steps of growing saidcells in a growth medium, through at least two passages, said medium.[.an.]. .Iadd.a modified .Iaddend.MEM .[.zinc option type.]. mediumhaving about the following amino acid profile:

    ______________________________________                                        Item         Amount (milligrams per liter)                                    ______________________________________                                        L alanine    8.9                                                              L asparagine 75.0                                                             L aspartic acid                                                                            13.0                                                             L glutamic acid                                                                            14.7                                                             L glycine    7.5                                                              L proline    11.5                                                             L serine     52.5                                                             L arginine HCl                                                                             196.52                                                           L cystine    37.2                                                             L glutamine  584.0                                                            L histidine HCl                                                                            65.0                                                             L isoleucine 80.88                                                            L leucine    160.88                                                           L lysine     112.38                                                           L methionine 23.30                                                            L phenyl-alanine                                                                           50.15                                                            L threonine  74.18                                                            L tryptophan 15.61                                                            L valine     71.4                                                             L tyrosine   45.9                                                             ______________________________________                                    


12. A process as defined in claim 11 wherein said medium compriseseffective amounts of additional nutrients as follows:biotin folic acidThyroxine Insulin Hydrocortisone Essential Fatty Acids Vitamin A VitaminE Surfactants
 13. A process as defined in claims 11 or 12, whereintransfer of cells from one passage to another is primarily effected byuse of physical means and without use of cell-harming quantities ofproteolytic enzymes.